Decolorization of the Textile Dyes by Newly Isolated

Decolorization of the textile dyes by newly isolatedbacterial strains

Kuo-Cheng Chen a,*, Jane-Yii Wu a, Dar-Jen Liou a, Sz-Chwun John Hwang b

a Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan, ROCb Department of Civil Engineering, Chung Hua University, Hsinchu, Taiwan, ROC

Received 7 January 2002; received in revised form 19 September 2002; accepted 24 September 2002

Abstract

Six bacterial strains with the capability of degrading textile dyes were isolated from sludge samples and mud lakes.

Aeromonas hydrophila was selected and identified because it exhibited the greatest color removal from various dyes.

Although A. hydrophila displayed good growth in aerobic or agitation culture (AGI culture), color removal was the best

in anoxic or anaerobic culture (ANA culture). For color removal, the most suitable pH and temperature were pH 5.5�/

10.0 and 20�/35 8C under anoxic culture (ANO culture). More than 90% of RED RBN was reduced in color within 8

days at a dye concentration of 3000 mg l�1. This strain could also decolorize the media containing a mixture of dyes

within 2 days of incubation. Nitrogen sources such as yeast extract or peptone could enhance strongly the

decolorization efficiency. In contrast to a nitrogen source, glucose inhibited decolorization activity because the

consumed glucose was converted to organic acids that might decrease the pH of the culture medium, thus inhibiting the

cell growth and decolorization activity. Decolorization appeared to proceed primarily by biological degradation.

# 2002 Elsevier Science B.V. All rights reserved.

Keywords: Aeromonas hydrophila ; Azo dyes; Anthraquinone dyes; Indigo dyes; Microbial decolorization

1. Introduction

The first synthetic dye, mauevin, was discovered

in 1856. Since then, over 100 000 dyes have been

generated worldwide with an annual production of

over 7�/105 metric tones. Synthetic dyes are

widely used in textile, paper, food, cosmetics and

pharmaceutical industries (Zollinger, 1987; Carliell

et al., 1995). The inefficiency in dyeing processes

has resulted in 10�/15% of unused dyestuff entering

the wastewater directly (Zollinger, 1987; Spadarry

et al., 1994). Color present in dye effluent gives a

straightforward indication of water being polluted,

and discharge of this highly colored effluent can

damage directly the receiving water. Furthermore,

it is difficult to degrade the mixtures of the

wastewater from textile industry by conventional

biological treatment processes, because their ratio

of BOD/COD is less than 0.3 (Chun and Yizhong,

1999). In some cases, traditional biological proce-

dures were combined with physical- or chemical-

* Corresponding author. Tel.: �/886-3-571-6249; fax: �/886-

3-571-3014.

E-mail address: [email protected] (K.-C. Chen).

Journal of Biotechnology 101 (2003) 57�/68

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0168-1656/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.

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treatment processes to achieve better decoloriza-tion (Vandevivere et al., 1998), but chemical or

physical�/chemical methods are generally costly,

less efficient and of limited applicability, and

produce wastes, which are difficult to dispose of.

As a viable alternative, biological processes have

received increasing interest owing to their cost

effectiveness, ability to produce less sludge, and

environmental benignity (Banat et al., 1996).Therefore, to develop a practical bioprocess for

treating dye-containing wastewater is of great

significance.

The effectiveness of microbial decolorization

depends on the adaptability and the activity of

selected microorganisms. Over the past decades,

many microorganisms are capable of degrading

azo dyes, including bacteria (Zimmerman et al.,1982; Haug et al., 1991; Sani and Banerjee, 1999),

fungi (Gold and Alic, 1993; Swamy and Ramsay,

1999; Balan and Monteiro, 2001; Novotny et al.,

2001), yeast (Martins et al., 1999), actinomycetes

(Zhou and Zimmermann, 1993) and algae (Dilek

et al., 1999). Most azo dyes are reduced anaero-

bically to the corresponding amines with cleavage

of azo bonds by bacterial azoreductase, but theyare difficult to degrade aerobically (Zimmerman et

al., 1982; Banat et al., 1996). Moreover, fungal

ligninolytic enzyme system (lignin peroxidase

(LiP), manganese peroxidase (MnP) and laccase)

might also be involved in the bio-oxidation of dyes

(Gold and Alic, 1993). However, the low pH

requirement (Swamy and Ramsay, 1999) for an

optimum activity of the enzymes and the longhydraulic retention time for complete decoloriza-

tion (Banat et al., 1996; Swamy and Ramsay,

1999) are the disadvantages of using fungi. In

addition, they may inhibit the growth of other

useful microorganisms. Thus, large-scale applica-

tions of fungal decolorization have been limited.

In general, the wastewater from textile industry

contains many various dyes. To gain a widespreadreception, the azo-degrading bacteria should ex-

hibit decolorizing ability for a wide range of dyes.

This study aimed to isolate some bacterial strains,

which possessed the ability to decolorize 24 kinds

of dyes, including azo, anthraquinone, and indigo

dyes. A bacterium displaying the greatest decolor-

izing ability was chosen for further study to

illustrate the factors influencing its efficiency. Inaddition, the major cause of the inhibition of

glucose on the reduction of azo dye was identified.

2. Materials and methods

2.1. Chemicals

Twenty-four dyes were used and chosen from

various types (azo, anthraquinone and indigo) of

important commercial dyes. Azo, anthraquinone

and indigo, containing various substituents such as

nitro and solfonic groups, are the major classes of

dyes with the greatest variety of colors. Acid Blue

74, Acid Orange 7, Acid Red 106, Direct Yellow 4

and Direct Yellow 12 were purchased from theSigma Chemical Company, MO, USA. The other

dyes (Acid Black 172, Acid Blue 264, Acid Yellow

42, Direct Black 22, Direct Orange 39, Direct Red

224, Direct Red 243, Direct Yellow 86, Reactive

Black NR, Reactive Black 5, Reactive Blue 160,

Reactive Blue 171, Reactive Blue 198, Reactive

Blue 222, Reactive Green 19, Reactive Red 120,

Reactive Red 141, Reactive Red 198 and ReactiveYellow 84) were obtained from Everlight Chemical

Industrial Co., Taoyuan, Taiwan. All other che-

micals were reagent grade.

2.2. Screening of decolorizers

Sludge samples were obtained from various

sources including the lake-mud in Tsing Hua

University (Hsinchu, Taiwan) and the sludge ofwastewater treatment plant in Chang Chun Petro-

chemical Co. (Miaoli, Taiwan). In order to obtain

a high-performance bacterial decolorizer, RED

RBN, the most commonly used dye, was first

chosen as the target for screening azo-degrading

bacteria. The mixed bacterial cultures from the

sludge samples were acclimated for 3 months, and

then served as the stock culture. The bacteria-isolating procedures and the test procedures later

used for each dye were carried out in a screening

medium (SM medium). The medium contained the

following components: yeast extract, 10 g; NaCl,

5.0 g in 1 l of distilled water with 0.1 g (except that

described else) of selected dye. Ten milliliter of the

K.-C. Chen et al. / Journal of Biotechnology 101 (2003) 57�/6858

stock culture was added to a 500 ml conical flaskcontaining 100 ml of SM medium and pH was

adjusted to 7.0. The isolates were cultured routi-

nely in an incubator at 30 8C. Next, the broth of

the decolorized flask culture was then transferred

to a fresh SM medium to screen strains that have

color removal ability. The screening procedures in

the liquid culture with SM medium were con-

ducted repeatedly until a decolorizing cultureappeared. An aliquot (0.1�/1 ml samples) of each

supernatant fluid of the isolated cultures was

spread on a SM agar medium, and then incubated

at 30 8C. Colonies surrounded by a decolorized

zone were selected. Isolates were then tested for

their color removal ability in a submerged culture.

Finally, six promising isolates were selected.

2.3. Dye assays and decolorizing cultures

The stock cultures for these isolates were pre-

cultured for 20 h at 30 8C by growing a singlecolony in an anoxic static condition. The same

initial cell concentrations of dye-degrading micro-

organisms were used to decolorize all the dyes.

Decolorization in an individual dye solution could

be seen visually, and was measured at its max-

imum adsorption wavelength (lmax) on culture

supernatants using a scanning spectrophotometer

(UV/vis, Shimadzu, Kyoto, Japan). To ensure thatthe pH change in dye solution did not influence

decolorization, the visible absorption spectra were

recorded between pH 4.0 and 11.0 and the pH did

not affect spectrum. Biomass concentration was

determined by dry cell weight after 24 h drying at

105 8C. All assays were conducted in triplicates

with uninoculated controls.

2.4. Analysis of color removal in the medium

containing mixture of dyes

All 24 dyes, each at a concentration of 50 mgl�1, were dissolved together in SM medium. The

mixture of dyes did not have a well-defined peak at

the visible absorption spectra. Therefore, the

detection of color level was made using the

American Dye Manufacturers Institute (ADMI)

Tristimulus Filter Method (Eaton et al., 1995).

2.5. Identification of selected azo dye-degrading

bacteria

Bacterial isolates with the greatest decoloriza-

tion abilities were first examined by Gram stain-

ing, and further identifications were performed by

the Culture Collection and Research Center, Food

Industry Research and Development Institute

(Hsinchu, Taiwan).

2.6. Decolorization at different culture conditions

The effects of the various culture conditions

such as agitation, aeration, anoxic state and

anaerobic state on decolorization of RED RBN

were examined owing to their various concentra-

tions of dissolved oxygen (DO). Agitation culture(AGI culture), the only culture at shaking condi-

tion, was operated in a rotary incubation shaker

running at 200 rpm. All the other cultures were

under a static condition with no shaking at all.

Anaerobic culture (ANA culture) was bubbled

with pure nitrogen only at the beginning until the

DO became zero, but anoxic culture (ANO

culture) had never been bubbled at all. Aerobicculture (AER culture) was maintained in a con-

tinuous aeration condition (airflow rate of 3 l

min�1). All the experiments were operated at

30 8C and pH 7.0 under a constant initial dye

concentration (RED RBN) of 50 mg l�1. The

concentration of cells, RED RBN, and DO were

monitored as a function of time.

2.7. Glucose analysis

Reducing sugar was analyzed and determined as

glucose by the DNS (3, 5-dinitrosalicylic acid)

method (Miller, 1959). The color tests were made

with 3 ml aliquots of reagent added to 3 ml

aliquots of sample in tubes. The reagent contained

1% dinitrosalicylic acid, 0.2% phenol, 0.05%

sodium sulfite, and 1% sodium hydroxide. Themixtures were heated for 15 min in a boiling water

bath, and then cooled and adjusted to ambient

temperature under running tap water. The color

intensities were measured in a scanning spectro-

photometer (UV/vis, Shimadzu, Kyoto, Japan) at

575 nm.

K.-C. Chen et al. / Journal of Biotechnology 101 (2003) 57�/68 59

Table 1

Decolorization of textile dyes by various bacteria

Type of dyes (C. I. Number) Chemical

structure of

dye

lmax (nm) Color removal (%) at initial concentration (100 mg l�1)

DEC1 DEC2 DEC3 DEC4 DEC5 DEC6

1 day 7 days 1 day 7 days 1 day 7 days 1 day 7 days 1 day 7 days 1 day 7 days

Azo

Acid Orange 7 (15510) Monoazo 485 799/2 1009/1 479/3 1009/2 389/1 1009/2 759/2 1009/3 449/2 1009/3 279/2 1009/2

Acid Red 106 (18110) Monoazo 533 709/2 1009/1 529/2 1009/1 629/5 1009/2 859/6 1009/1 459/5 1009/2 639/3 1009/3

Direct Orange 39 (40215) Monoazo 414 739/3 1009/1 509/2 1009/1 429/4 1009/1 759/2 1009/2 519/4 1009/5 399/5 1009/6

Reactive Red 198 (unpublished) Monoazo 515 659/1 1009/2 859/2 1009/2 869/3 1009/3 899/3 1009/1 829/3 1009/3 829/5 1009/3

Acid Yellow 42 (22910) Diazo 412 159/1 649/3 69/1 449/3 79/3 459/1 119/2 589/3 39/2 349/1 29/1 69/1

Direct Red 224 (unpublished) Diazo 520 59/1 669/3 179/2 499/3 159/4 699/5 129/2 569/2 89/2 579/2 99/1 329/2

Direct Red 243 (29315) Diazo 523 409/2 849/2 229/2 659/2 139/1 689/4 399/1 809/2 159/1 519/2 129/3 669/3

Direct Yellow 4 (24890) Diazo 396 469/2 1009/2 249/2 1009/2 199/2 839/3 609/2 1009/3 229/2 1009/2 149/1 1009/2



Reactive Black 5 (20505) Diazo 597 469/2 959/2 679/3 539/4 629/1 599/5 689/3 859/2 669/3 559/5 639/2 549/5

Reactive Blue 222 (unpublished) Diazo 613 639/2 1009/2 389/2 719/2 439/3 709/4 679/2 1009/1 399/4 649/2 459/5 769/2

Reactive Red 141 (unpublished) Diazo 532 509/2 879/2 179/2 799/3 139/1 829/6 469/2 829/2 109/2 649/6 59/2 399/3

Reactive Red 120 (25810) Diazo 512 419/1 829/2 169/2 669/2 129/2 629/2 409/4 809/5 119/2 599/2 109/1 809/5

Direct Black 22 (35435) Polyazo 482 429/2 699/2 259/2 649/2 199/2 659/3 159/2 459/2 79/2 349/2 59/2 239/3

Acid Black 172 (unpublished) Azo 571 139/1 519/3 89/1 389/4 99/1 409/5 99/1 359/6 29/1 349/2 0 0

Reactive Blue 160 (unpublished) Azo 616 659/1 1009/1 609/2 1009/3 589/1 1009/4 679/2 1009/2 659/2 1009/4 629/2 1009/4



Reactive Black NR (unpublished) Azo 598 819/2 1009/2 789/3 1009/2 789/1 1009/2 839/3 1009/2 779/5 1009/3 779/5 1009/2

Reactive Green 19 (unpublished) Azo 630 439/1 839/2 149/2 709/3 109/3 659/2 509/2 779/5 79/2 589/2 209/1 709/3

Reactive Yellow 84 (unpublished) Azo 411 459/2 639/3 199/2 629/2 159/2 609/3 439/4 559/2 149/2 509/5 119/2 449/1

Anthraquinone

Acid Blue 264 (unpublished) Anthraquinone 608 459/2 809/1 279/2 779/3 239/2 689/2 469/3 679/2 259/2 659/2 39/1 199/2

Indigoid

Acid Blue 74 (73015) Indigoid 609 609/2 849/3 509/4 819/3 469/5 859/3 409/1 779/3 309/3 729/5 269/2 709/3

The names of all the dyes above are recognized by the Color Index.

K.-C

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3. Results and discussion

3.1. Isolation and identification

White and pink colonies surrounded by an

almost decolorized zone were isolated and then

tested for color removal capability using sub-

merged cultures. Among these colonies, six of

them with the highest decolorization ability in

SM medium, designated as DEC1-6, were selectedfor a further study.

Decolorization of various dyes by the growing

cells of the six isolates were shown in Table 1.

Among the 24 dyes, Acid Orange 7, Acid Red 106,

Direct Orange 39, Direct Yellow 4, Direct Yellow

12, Reactive Black NR, Reactive Blue 160 and

Reactive Red 198 were reduced completely by all

the strains, DEC1�/6, while Reactive Blue 198 (5�/

22%) and Acid Black 172 (0�/51%) were reduced

only slightly even after 7 days of incubation. The

effectiveness of all the six isolates in decolorizing

these 24 dyes may depend on the structure and

complexity of the dyes, particularly on the nature

and position of substituent in the aromatic rings

and the resulting interactions with the azo bond

(Zimmerman et al., 1982; Sani and Banerjee,1999). However, no clear relationship can be

observed between the position of substituent in

the aromatic rings from published structure of dye

and the decolorization efficiency using dye-degrad-

ing microorganisms in this study, except that most

monoazo dyes tested had color removal higher

than the diazo dyes and anthraquinone dyes tested

under the same initial biomass. The differentefficiency may be due to the number of azo groups.

Similar observation was obtained on the investiga-

tion of the degradability in different structures of

azo dyes by Phanerochaete chrysosporium (Pod-

gornik et al., 1999).

On the other hand, Table 1 also shows that the

decolorization rate of the six isolates were

DEC1�/DEC4�/DEC2X/DEC3�/DEC5�/

DEC6 after 1 day of incubation under the same

initial cell concentrations. However, if we want to

consider an isolate favorable for development of a

practical bioprocess for decolorization, the deco-

lorization rate is very important. Therefore, sev-

eral biochemical and physiological investigations

were conducted to identify the best strain, DEC1.

The strain was identified as Aeromonas hydrophila

according to the GN microplate (Biolog, CA,

USA), API 20E (BioMerieux SA, Marcy l’etoile,

France), API 50 CHE (BioMerieux) and partial

sequencing of 16S rRNA gene. The characteriza-

tion of strain DEC1 was summarized in Table 2.

From phylogenetic analysis based on 16S rRNA

sequence, strain DEC1 was also identified as a

strain that is most related to A. hydrophila .

Table 2

Biochemical and physiological profiles of strain DEC1

Characteristics A. hydrophila (DEC1)

Morphology Rod

Motile �/

Gram staining �/

Aerobic growth �/

Anaerobic growth �/

Nitrate reduction �/

Catalase �/

Gas from glucose �/

H2S from cysteine �/

Acetoin from glucose �/

Indole production �/

Acid from glucose �/

Arginine dihydrolase �/

b-galactosidase �/

Cytochrome oxidase �/

Hydrolysis of

Esculin �/

Gelation �/

Assimilation of

Adipate �/

Arabinose �/

Citrate �/

Gluconate �/

Glucose �/

Malate �/

Maltose �/

Mannitol �/

Mannose �/

N -acetyl-glucosamine �/

Phenyl-acetate �/

Acid from

Arabinose �/

Maltose �/

Mannitol �/

Xylose �/


In addition, large quantities of RED RBN and

Remazol Black B are now used in textile and

dyestuff industries in Taiwan. RED RBN and

Remazol Black B are denoted as Reactive Red 198

and Reactive Black 5 in Color Index, respectively.

Thus, RED RBN and Remazol Black B were

chosen as the target dyes for further study on

microbial characteristics and the causes of degra-

dation by the representative strain DEC1, A .

hydrophila .

3.2. Characteristics of microbial decolorization

Bacterial degradation of azo dyes is often an

enzymatic reaction linked to anaerobiosis, because

it is inhibited by oxygen that is in competition with

the azo group as the electron receptor in the

oxidation of the reduced electron carrier, i.e.

NADH (Wuhrmann et al., 1980; Zimmerman et

al., 1982; Banat et al., 1996). Seldom are bacteriaable to decolorize azo compounds in the presence

of oxygen (Wuhrmann et al., 1980). Although the

strong oxygen effect on bacterial decolorization

has been proved definitely, the quantitative corre-

lation between DO and color removal has seldom

been reported. A. hydrophila was propagated in

Fig. 1. Effect of various culture conditions on decolorization

by A. hydrophila at 30 8C in SM medium containing 50 mg l�1

RED RBN. (j) AER culture (air flow rate of 3 l min 1�1); (m)

AGI culture (rotary agitation at 200 rpm); (') ANO culture

(no aeration, no agitation); (^) ANA culture (gassing the flasks

with pure nitrogen before static culture).

Fig. 2. Effect of pH on color removal of RED RBN by A.

hydrophila in SM medium at 30 8C under ANO culture. Initial

dye concentration: 100 mg l�1. (j) 1 day of cultivation; (m) 2

days of cultivation; (') 3 days of cultivation.


SM medium using AER, ANO, ANA, or AGIculture to observe cell growth, DO concentration

and decolorization (Fig. 1). In both AGI and AER

cultures, the presence of oxygen would normally

inhibit the activity of decolorization, resulting in a

low efficiency of color removal with A. hydrophila .

As a matter of fact, AGI or AER culture was only

run for 1.5 days and then switched to anoxic static

condition. Color disappeared due to the DOconcentration dropping to almost zero. The above

results suggest that decolorization of azo dye

would not take place at a DO concentration higher

than 0.45 mg l�1 and a slight increase in cell mass

at the initial stage would enhance the efficiency of

color removal (Fig. 1). Therefore, the results

indicated that the decolorization by A. hydrophila

was very sensitive to DO level. To achieve aneffective color removal, agitation and aeration

should be avoided.

Fig. 2 shows that the suitable pH for decolor-

ization of RED RBN ranged from 5.5 to 10.0 with

a sharp change toward both ends of the pH range.

At the two extreme pH values (i.e. pH 4.5 and

11.0), a strong negative effect occurred signifi-

cantly on the growth of bacteria and the stabiliza-tion of pH. These results show that decolorization

of various types of dyes with A. hydrophila

occurred over an extensive range of pH. In other

words, they are favorable for developing a prac-

tical bioprocess for a dye-containing wastewater.

Additionally, when the initial pH of the culture

was at 4.5, the cell mats were deeply colored by

adsorbed dyes only. The adsorption of dye on thecell surface may be related to the mechanism of

charge neutralization. Normally, the dyes tested

are negatively charged. In contrast, the cells in

solution tend to possess relatively positive charges

at lower pH. Thus, the cells may have relatively

higher affinity for the dyes.

Whether RED RBN was used as a substrate for

A. hydrophila , a proper color removal, specificdecolorization rate and cell growth under ANO

culture was observed in the range of 20�/35 8C(data not shown). The low color removal at a

temperature beyond 35 8C may be attributed to

the thermal deactivation of the decolorization

enzymes and the low biomass. According to the

above results, the following decolorization experi-

ments using A. hydrophila were performed at

30 8C and pH 7.0 under ANO culture.

To determine the maximum RED RBN con-

centration tolerated by A. hydrophila , experiments

with different initial dye concentrations (1000�/

8000 mg l�1) were performed. The decolorization

efficiency was above 90% for initial dye concen-

tration less than 3000 mg l�1 after 8 days

cultivation, but it decreased with further increase

in dye concentration. When the dye concentration

was as high as 8000 mg l�1, almost 60% of the dye

was removed after 10 days of cultivation (data not

shown). This means that an acceptable high color

removal can be achieved by the A. hydrophila

strain in an extensive range of azo dye concentra-

tions. In addition, a substrate inhibition effect was

observed at dye concentrations higher than 3000

mg l�1. Reduction in color removal and cell

growth may result from the toxicity of dyes to

bacteria through the inhibition of metabolic activ-

ities. Azo dyes generally contain one or more

sulphonic-acid groups on aromatic rings, which

might act as detergents to inhibit the growth of

microorganisms (Wuhrmann et al., 1980). On the

other hand, it was also reported that dyes were the

Fig. 3. Effect of various nitrogen sources on decolorization of

RED RBN by A. hydrophila at 30 8C under ANO culture.

Initial nitrogen sources concentration: 10 g l�1. (I) blank

(without any nitrogen); (j) peptone; (m) tryptone; (')

monosodium glutamate; (%) meat extract; (") beef extract;

(k) urea; (^) yeast extract.


inhibitors for nucleic acid synthesis (Ogawa et al.,

1988) or for cell growth (Ogawa et al., 1989).

Furthermore, the color removal exhibited a

growth-associated pattern (data not shown). Themaximum cell growth yield was about 1.2�/1.6 and

0.7�/1.0 g l�1 for dye concentrations between 1000

and 3000 and 4000�/8000 mg l�1, respectively. Our

works on the association of growth (kinetic para-

meters) and decolorization by A. hydrophila are

now in progress. Similar results were obtained

using Remazol Black B instead of RED RBN.

3.3. Effects of nitrogen sources on decolorization

Fig. 3 shows the influence of various organic

nitrogen sources on the efficiency of decolorization

of RED RBN by A. hydrophila . Decolorizationwith peptone or yeast extract was very effective, so

the dye concentration decreased quickly, resulting

in 90% color removal within 2 days of cultivation.

In addition to the organic nitrogen sources, the

inorganic nitrogen sources such as KNO3,

NaNO3, NaNO2, NH4Cl, (NH4)2SO4 were also

selected for decolorization. Similar performances

were observed with control flasks (without any

nitrogen source) but resulting in around 10�/15%

color removal after 6 days cultivation (data not

shown). The results clearly indicate that decolor-

ization of RED RBN by A. hydrophila was greatly

affected by the addition of various nitrogen

sources. The metabolism of yeast extract is con-

sidered essential to the regeneration of NADH

that acts as the electron donor for the reduction of

azo bonds (Carliell et al., 1995). Between these two

nitrogen sources, yeast extract was finally chosen

as a part of culture medium for further experi-

ments because yeast extract is cheaper than

peptone. Similar results were obtained using

Remazol Black B instead of RED RBN.

It had also been found that increasing yeast

extract concentrations (from 0 to 10 g l�1) resulted

in higher decolorization rates, and the decoloriza-

tion rates reached a plateau as yeast extract was

higher than 8 g l�1. However, the color removal

(�/90%) was not enhanced significantly by the

increase in yeast extract from 8 to 10 g l�1 after 1

Fig. 4. Time courses of growth and decolorization of mixture dyes by A. hydrophila at 30 8C under static culture (initial pH 7.0). (m),

Dye concentration; (k), biomass.


day of incubation (data not shown). Therefore,

yeast extract at a concentration of 8 g l�1 as

nitrogen source for decolorization of A. hydrophila

was added in further experiments.

3.4. Decolorization of mixed dyes

Dyes of different structures are often used in the

textile processing industry, and, therefore, the

effluents from the industry are markedly variablein composition. A nonspecific biological process

may be vital for treatment of the textile effluents

containing a mixture of dyes. The rate of decolor-

ization was very fast and the color removal was

almost 90% within 2 days of cultivation, followed

by an insignificant change in decolorization for the

next 10 days (Fig. 4). Fig. 4 also displayed a

growth-associated pattern on color removal.According to the reports (Knapp and Newby,

1995; Sani and Banerjee, 1999) decolorization of

dyes by bacteria can be due to adsorption to

microbial cells or to biodegradation. In adsorp-

tion, examination of the absorption spectrum will

reveal that all peaks decrease approximately in

proportion to each other. If the dye removal is

attributed to biodegradation, either the major

visible light absorbance peak will completely

disappear or a new peak will appear. Dye adsorp-

tion can also be judged clearly by inspecting the

cell mats. Cell mats become deeply colored be-

cause of adsorbing dyes, whereas those retaining

their original colors are accompanied by the

occurrence of biodegradation. The absorbance

peak at 515 nm (A point) disappeared completely

after 7 days cultivation (Fig. 5a). As seen in Fig.

5b, there was a significant decrease in color

intensity or in the peak absorbance at 306, 370

and 597 nm (Points B, C and D, respectively).

Moreover, as the RED RBN and Remazol Black

B were removed, the A. hydrophila strain remained

colorless. A similar result was also observed in a

Fig. 5. Variation in UV�/visible spectra of various dye solu-

tions after decolorizing cultivation with A. hydrophila . (*/)

Original dye solution; (� � �) decolorized dye solution.

Fig. 6. Effect of glucose concentration on decolorization of

RED RBN by A. hydrophila in SM medium at 30 8C under

ANO culture ( initial pH 7.0). Initial glucose concentration, (I)

0 g l�1; (j) 0.15 g l�1; (m) 1.25 g l�1; (") 10.0 g l�1.


mixture of dyes. Consequently, according to theabove results, the color removal by A. hydrophila

strain might be largely attributed to biodegrada-

tion, and the biosorption onto the bacterial

surfaces was not significant.

3.5. Effect of glucose on the degradation of the

mixed dyes

Glucose has been added to enhance the decolor-ization performance of biological systems in some

studies (Haug et al., 1991; Carliell et al., 1995;

Kapdan et al., 2000). However, others reported

that glucose inhibited the decolorizing activity

(Chung et al., 1978; Knapp and Newby, 1995).

The variability may be due to the different

microbial characteristics. In this study, various

concentrations of glucose (0�/10 g l�1) were firstevaluated for decolorization of RED RBN by A.

hydrophila under ANO culture (Fig. 6). Fig. 6

clearly indicates that glucose concentration of

higher than 0.15 g l�1 inhibited appreciably the

azo reduction of azo dye by A. hydrophila . In

addition, the color of the cell surface became red,

and the color removal, decolorization rate and

biomass decreased significantly with increasingglucose concentration.

Fig. 6 depicts that after 1 day cultivation, only

1.0�/1.9 g l�1 of glucose was consumed in the

medium supplemented with the glucose concentra-

tion at a range of 1.25�/10 g l�1, and the pH of the

media dropped from 7.0 to 4.7�/5.0, followed by a

relatively stable pH value for the next 2 days.

Obviously, this low pH range (4.7�/5.0) had asignificantly negative effect on the growth of

bacteria, so the decolorization of azo groups was

inhibited. These results are also in good agreement

with those found at lower pH as aforementioned

(Fig. 2). Moreover, while the pH of the medium

was adjusted to 7.0 by adding aseptic NaOH after

3 days cultivation, it is worthy of note that color

removal of RED RBN in this culture was in-creased from 25 to 90% within 2 days (data not

shown), and the color of cell surface changed

visually from red to white (original color of the

cell). According to above results, it is inferred that

as consumption of glucose concentration in-

creased, the rate of accumulation of organic acids

in the medium was also increased. The growth and

decolorization of A. hydrophila were inhibited at

lower pH in the medium.

To confirm that glucose inhibited the decolor-

ization activity and the cell growth was due mainly

to the lower pH that was, in part, caused by the

consumed glucose or converted organic acids,

phosphate buffer was added into the medium to

provide pH control during growth and dye deco-

lorization (Fig. 7). The pH of the culture supple-

mented with phosphate buffer dropped much less

than that without buffer because the phosphate

buffer was proved to provide a good pH control as

well as high decolorization activity and cell growth

of A. hydrophila . The results clearly show that the

inhibition of cell growth and bacterial decoloriza-

tion of azo dye by glucose was attributed to the

reduced pH in the surrounding medium through a

Fig. 7. Effect of glucose concentration on decolorization of

RED RBN by A. hydrophila in SM supplemented with or

without buffer at 30 8C under ANO culture after 2 days

cultivation (initial pH 7.0). (j) Supplemented with phosphate

buffer; (I) without buffer.


biological conversion to organic acids. This de-monstrates the importance of pH control to

decolorization if some very biodegradable carbon

sources were present in dye wastewater. Many

aspects of the mechanisms involved in the inhibi-

tion of decolorization by glucose are still scarcely

known.

4. Conclusions

The results indicate that utilization of A. hydro-

phila was suitable for the decolorization of dyes

(RED RBN and Remazol Black B) in the presence

of a nitrogen source such as yeast extract. Cer-

tainly, the use of yeast extract as a nitrogen source

for cell growth would be of low economic effi-ciency in the application of industrial treatment

plant. In order to enhance process efficiency, the

search for cheaper supplementary nitrogen sources

would be essential in future works. In contrast to

nitrogen sources, glucose showed inhibitory effects

on the cell growth and the decolorization activity.

Additionally, to ensure an effective azo dye

decolorization with A. hydrophila required arigorous control of the DO concentration (B/

0.45 mg l�1) in the biological process. High dye

concentrations (�/3000 mg l�1) might have a

toxic effect on the isolate. This strain could also

decolorize synthetic effluent containing a mixture

of different dyes. That is applicable to a wide

variety of individual dyes and mixture of dyes.

Acknowledgements

The authors acknowledge the financial support

of National Science Council of Republic of China

under Grant No. NSC-89-2211-E-007-005.

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Decolorization of the Textile Dyes by Newly Isolated